Project Summary/Abstract The expansion of follicular helper (Tfh) cells and related subpopulations is correlated with the severity of human lupus disease and is shared by all lupus-prone mouse models. However, the mechanisms for the expansion of Tfh cells in lupus are largely unknown. Thus, the objective of this proposal is to shed new light on the mechanisms that are responsible for Tfh cell expansion in lupus using innovative cellular and molecular approaches based on the obtained data from the B6.Sle1.Sle2.Sle3 triple congenic (TC) murine model of lupus. Accordingly, we have identified genetic, as well as microbial and metabolic determinants that modulate lupus Tfh cell expansion. Transferring the perturbed gut microbiota of lupus mice into mouse host crucially contributed to Tfh cell expansion. Additionally, an altered tryptophan (Trp) metabolism also enhanced Tfh cell expansion, which was reduced by manipulating dietary Trp levels in mice. Finally, inhibiting glycolysis with 2-deoxi-D-glucose (2DG), or glutaminolysis with 6-Diazo-5-oxo-L-norleucine (DON) functionally diminished lupus Tfh cell differentiation. Our hypothesis is that the expansion of Tfh cells implicated in lupus integrates cues from susceptibility genes, dysregulated microbiome and environmental metabolites. In support of this hypothesis, our data demonstrate that TC Tfh cells possess a novel metabolic gene signature, potentially driving the autoreactive Tfh cell expansion directed by a unique metabolic flux. Thus, we propose combining the analyses of Tfh cell transcriptome and metabolome in response to variations of the aforementioned cues as a means to elucidate the involved molecular mechanisms, singly or in combination, impacting the fate of Tfh cells in lupus. We also propose to investigate the effects of genetic and environmental determinants functionally affecting Tfh cells on two B cell subsets, CD11c+Tbet+ B cells (ABCs) and IgA+ B cells. To proceed, the following Specific Aims are proposed. 1. To explore the mechanisms by which lupus susceptibility genes modulate the Tfh cell transcriptome and metabolome in the two unrelated TC and (NZW x BXSB.Yaa)F1 lupus models. 2. To identify the impact of environmental factors on the Tfh transcriptome and metabolome in TC mice. And 3. To elucidate the molecular mechanisms implicated in the expansion of Tfh cells in lupus patients. Obtained results from these mechanistic experiments will be the first in-depth investigation of Tfh cell expansion directed by environmental and genetic determinants, which may be reprogrammed for a sustainable therapeutic strategy to perhaps improve lupus patients' health.